Influence of carbonization conditions on luminescence and gene delivery properties of nitrogen-doped carbon dots

Carbon dots (CDs) have been intensively investigated due to their unique photoluminescence (PL) properties that are improved through surface passivation with nitrogen-containing groups. Recently, gene delivery applications emerged as passivation of CDs may yield positively charged nanoparticles that can interact with negatively charged nucleic acids. However previous work in the field focused on the use of high molecular weight polyamines for CD passivation, posing the problem of the separation of nanoparticles from residual polymer that is harmful to cells. In this work, cationic CDs were prepared by pyrolysis of citric acid/bPEI600 (1/4, w/w) so unreacted low molecular weight reagents could be conveniently eliminated by extensive dialysis. Various reaction conditions and activation modes were evaluated and eleven CDs that exhibited superior solubility in water were produced. All the nanoparticles were characterized with respect to their physical, optical and PL properties and their ability to deliver plasmid DNA to mammal cells was evaluated. Despite their similar physical properties, the CDs displayed marked differences in their gene delivery efficiency. CDs produced under microwave irradiation in a domestic oven were revealed to be superior to all the other nanoparticles produced in this study and compared to the gold standard transfection reagent bPEI25k, with an optimal CD/pDNA w/w ratio that was significantly down shifted, as was the associated cytotoxicity.

Carbon dots prepared from citric acid and bPEI600 using various activation modes were evaluated as gene delivery reagents.     

Sustained delivery and expression of DNA encapsulated in polymeric nanoparticles

Sustained release polymeric gene delivery systems offer increased resistance to nuclease degradation, increased amounts of plasmid DNA (pDNA) uptake, and the possibility of control in dosing and sustained duration of pDNA administration. Furthermore, such a system lacks the inherent problems associated with viral vectors. Biodegradable and biocompatible poly(DL-lactide-co-glycolide) polymer was used to enacapsulate pDNA (alkaline phosphatase, AP, a reporter gene) in submicron size particles. Gene expression mediated by the nanoparticles (NP) was evaluated in vitro and in vivo in comparison to cationic-liposome delivery. Nano size range (600 nm) pDNA-loaded in poly(DL-lactide-co-glycolide) polymer particles with high encapsulation efficiency (70%) were formulated, exhibiting sustained release of pDNA of over a month. The entrapped plasmid maintained its structural and functional integrity. In vitro transfection by pDNA-NP resulted in significantly higher expression levels in comparison to naked pDNA. Furthermore, AP levels increased when the transfection time was extended, indicating sustained activity of pDNA. However, gene expression was significantly lower in comparison with standard liposomal transfection. Seven days after i.m. injections in rats, naked pDNA and pDNA-NP were found to be significantly more potent (1-2 orders of magnitude) than liposomal pDNA. Plasmid DNA-NP treatment exhibited increased AP expression after 7 and 28 days indicating sustained activity of the NP.     

Localization of exogenous DNA to mitochondria in skeletal muscle following hydrodynamic limb vein injection

Mitochondrial genetic disorders are a major cause of mitochondrial diseases. It is therefore likely that mitochondrial gene therapy will be useful for the treatment of such diseases. Here, we report on the possibility of mitochondrial gene delivery in skeletal muscle using hydrodynamic limb vein (HLV) injection. The HLV injection procedure, a useful method for transgene expression in skeletal muscle, involves the rapid injection of a large volume of naked plasmid DNA (pDNA) into the distal vein of a limb. We hypothesized that the technique could be used to deliver pDNA not only to nuclei but also to mitochondria, since cytosolic pDNA that is internalized by the method may be able to overcome mitochondrial membrane. We determined if pDNA could be delivered to myofibrillar mitochondria by HLV injection by PCR analysis. Mitochondrial toxicity assays showed that the HLV injection had no influence on mitochondrial function. These findings indicate that HLV injection promises to be a useful technique for in vivo mitochondrial gene delivery.     

共聚物P85和微泡造影剂对小鼠骨骼肌超声介导基因转染的影响

目的 探讨共聚物P85、微泡造影剂和超声在质粒DNA对小鼠骨骼肌基因转染中的影响.方法 应用共聚物P85、微泡造影剂Optison与DNA混合后直接小鼠胫前肌(TA)注射,并辐照超声.1周后取出胫前肌并快速冰冻切片,荧光显微镜计数表达GFP转染的肌纤维数,HE染色评价肌肉损伤情况.结果 共聚物P85和微泡造影剂Optison均可促进质粒DNA的基因转染(P<0.01,P<0.05).辐照超声可使P85介导的基因转染效率显著提高(P<0.01),但对微泡造影剂介导的基因转染却无显著提高(P>0.05),并且P85所介导的基因转染效率高于微泡造影剂介导的基因转染效率(P<0.01).微泡造影剂和P85耦合并辐照超声可使质粒的基因转染效率显著提高,与所有各组的差异有统计学意义(P<0.01).同时辐照超声显著增加含微泡造影剂组骨骼肌的损伤面积(P<0.01).结论 共聚物P85和微泡造影剂可介导质粒DNA的基因转染,辐照超声对其有促进作用,三者联合应用具有协同作用.     

Reducible poly(amido ethylenediamine) for hypoxia-inducible VEGF delivery.

Delivery of the hypoxia-inducible vascular endothelial growth factor (RTP-VEGF) plasmid using a novel reducible disulfide poly(amido ethylenediamine) (SS-PAED) polymer carrier was studied in vitro and in vivo. In vitro transfection of primary rat cardiomyoblasts (H9C2) showed SS-PAED at a weighted ratio of 12:1 (polymer/DNA) mediates 16 fold higher expression of luciferase compared to an optimized bPEI control. FACS analysis revealed up to 57+/-2% GFP positive H9C2s. The efficiency of plasmid delivery to H9C2 using SS-PAED was found to depend upon glutathione (GSH) levels inside the cell. SS-PAED mediated delivery of RTP-VEGF plasmid produced significantly higher levels of VEGF expression (up to 76 fold) under hypoxic conditions compared to normoxic conditions in both H9C2 and rat aortic smooth muscle cells (A7R5). Using SS-PAED, delivery of RTP-VEGF was investigated in a rabbit myocardial infarct model using 100 mug RTP-VEGF. Results showed up to 4 fold increase in VEGF protein expression in the region of the infarct compared to injections of SS-PAED/RTP-Luc. In conclusion, SS-PAED mediated therapeutic delivery improves the efficacy of ischemia-inducible VEGF gene therapy both in vitro and in vivo and therefore, has potential for the promotion of neo-vascular formation and improvement of tissue function in ischemic myocardium.     

Dose response in rodents and nonhuman primates after hydrodynamic limb vein delivery of naked plasmid DNA

The efficacy of gene therapy mediated by plasmid DNA (pDNA) depends on the selection of suitable vectors and doses. Using hydrodynamic limb vein (HLV) injection to deliver naked pDNA to skeletal muscles of the limbs, we evaluated key parameters that affect expression in muscle from genes encoded in pDNA. Short-term and long-term promoter comparisons demonstrated that kinetics of expression differed between cytomegalovirus (CMV), muscle creatine kinase, and desmin promoters, but all gave stable expression from 2 to 49 weeks after delivery to mouse muscle. Expression from the CMV promoter was highest. For mice, rats, and rhesus monkeys, the linear range for pDNA dose response could be defined by the mass of pDNA relative to the mass of target muscle. Correlation between pDNA dose and expression was linear between a threshold dose of 75 μg/g and maximal expression at approximately 400 μg/g. One HLV injection into rats of a dose of CMV-LacZ yielding maximal expression resulted in an average transfection of 28% of all hind leg muscle and 40% of the gastrocnemius and soleus. Despite an immune reaction to the reporter gene in monkeys, a single injection transfected an average of 10% of all myofibers in the targeted muscle of the arms and legs and an average of 15% of myofibers in the gastrocnemius and soleus.     

Smart and cationic poly(NIPA)/PEI block copolymers as non-viral vectors: in vitro and in vivo transfection studies

In this study, in vitro and in vivo transfection of temperature-sensitive, polycationic poly(N-isopropylacrylamide) and polyethyleneimine copolymers (poly(NIPA)/PEI25L) were performed. Copolymer and copolymer-plasmid DNA (pDNA) complexes were positively charged as + 7.6 and + 12.8, respectively. Gel retardation assay confirmed good complex formation and release of plasmid DNA in response to temperature and pH. Cytotoxicity tests showed at least 80% smooth muscle cell (SMC) viability. The uptake of the complexes by SMCs was quite high; however, the best gene expression efficiency achieved with the copolymeric vectors was about 30% with the complex prepared with a polymer:plasmid ratio of 6. Gene expression efficiency was enhanced up to 50% by changing the temperature from 37 degrees C to 28 degrees C. Preliminary in vivo studies were performed above and below lower critical solution temperature (LCST) in lung, heart, liver, kidney, muscle and also subcutaneously in 5 week-old mice. The gene expression ratio was higher in lung, tibial muscle and subcutaneously than in other tissues (heart, liver and kidney) above LCST. Then, temperature decrease caused an increase in the amount of gene expression in tibial muscle and subcutaneously, revealing the contribution of temperature-sensitivity on DNA release and gene expression.     

A sterically stabilized immunolipoplex for systemic administration of a therapeutic gene

A sterically stabilized immunolipoplex (TsPLP), containing an antitransferrin receptor single-chain antibody fragment (TfRscFv)-PEG molecule, has been developed to specifically and efficiently deliver a therapeutic gene to tumor cells. A postcoating preparation strategy was employed in which a DNA/lipid complex (lipoplex) was formed first and then sequentially conjugated with PEG and TfRscFv. The complex prepared by this method was shown to be superior in ability to deliver genes to tumor cells than when prepared by a common precoating strategy, in which DNA is mixed with TfRscFv-PEG conjugated liposome. Using prostate cancer cell line DU145, a comparison was made between the in vitro and in vivo gene delivery efficiencies of four complexes, Lipoplex (LP), PEG-Lipoplex (PLP), TfRscFv-PEG-Lipoplex (TsPLP) and our standard TfRscFv-Lipoplex (TsLP). In vitro, the order of transfection efficiency was TsLP>LP approximately TsPLP>PLP. However, in vivo the order of transfection efficiency, after systemic administration via the tail vein, was TsPLP>TsLP>LP or PLP with TsPLP-mediated exogenous gene expression in tumor being two-fold higher than when mediated by TsLP. This suggests that the in vitro transfection efficiency of TsPLP was not indicative of its in vivo efficiency. In addition, it was found that the level of exogenous gene expression in the tumor mediated by TsPLP was higher than that mediated by TsLP and did not decrease over the time. More importantly, high exogenous gene expression in tumor, but low expression in liver, was observed after an i.v. delivery of TsPLP carrying either the GFP reporter gene or the p53 gene, indicating that tumor preferential targeting was maintained by this complex in the presence of PEG. These findings show that incorporation of PEG into our targeted lipoplex results in a more efficient delivery of the complex to the tumor cells, possibly by inhibiting the first pass clearance observed with non-PEG containing liposomes. Therefore, these data demonstrate that TsPLP is a improvement over our previously established tumor targeted gene delivery complex for systemic gene therapy of cancer.     

Microbubble ultrasound improves the efficiency of gene transduction in skeletal muscle in vivo with reduced tissue damage

Intramuscular injection of naked plasmid DNA is a safe approach to the systemic delivery of therapeutic gene products, but with limited efficiency. We have investigated the use of microbubble ultrasound to augment naked plasmid DNA delivery by direct injection into mouse skeletal muscle in vivo, in both young (4 weeks) and older (6 months) mice. We observed that the albumin-coated microbubble, Optison (licensed for echocardiography in patients), significantly improves the transfection efficiency even in the absence of ultrasound. The increase in transgene expression is age related as Optison improves transgene expression less efficiently in older mice than in younger mice. More importantly, Optison markedly reduces muscle damage associated with naked plasmid DNA and the presence of cationic polymer PEI 25000. Ultrasound at moderate power (3 W/cm2 1 MHz, 60 s exposure, duty cycle 20%), combined with Optison, increases transfection efficiency in older, but not in young, mice. The safe clinical use of microbubbles and therapeutic ultrasound and, particularly, the protective effect of the microbubbles against tissue damage provide a highly promising approach for gene delivery in muscle in vivo.     

Artery wall binding peptide-poly(ethylene glycol)-grafted-poly(L-lysine)-based gene delivery to artery wall cells.

Artery wall binding peptide (AWBP; Cys-Gly-Arg-Ala-Leu-Val-Asp-Thr-Leu-Lys-Phe-Val-Thr-Gln-Ala-Glu-Gly-Ala-Lys), a specific targeting peptide, was conjugated to poly(ethylene glycol)-grafted-poly(L-lysine) (PEG-g-PLL) to enhance the gene transfer to artery wall cells. AWBP-PEG-PLL was synthesized by the reaction between the vinylsulfone group of PEG-g-PLL and the thiol group of cysteine in AWBP. 1H-NMR analysis confirmed the composition of the obtained polymer and indicated that four mol of AWBP were reacted to one mole of VS-PEG-PLL. The particles of AWBP-PEG-PLL/pDNA complexes were determined spherical with a size of approximately 100 nm by dynamic light scattering (DLS) and atomic force microscopy (AFM). Agarose gel retardation assay indicated that AWBP-PEG-PLL was able to condense plasmid DNA and reach complete complexation at and above a charge ratio 1/1 (+/-). Transfection efficiency of AWBP-PEG-PLL/pDNA complexes was 150-180 times higher than that of control systems, such as PEG-g-PLL/pDNA and PLL/pDNA, in both bovine aorta endothelial cells and smooth muscle cells. Luciferase activities of AWBP-PEG-PLL depended on the amount of free AWBP, while those of the control carriers such as PLL and PEG-g-PLL were not affected by free AWBP. These results supported that gene transfer of AWBP-PEG-PLL/pDNA complexes to bovine aorta wall cells was mediated by specific artery wall cell receptor-mediated endocytosis.     

Successful in vivo tumor targeting of prostate-specific membrane antigen with a highly efficient J591/PEI/DNA molecular conjugate

We have utilized a novel polyethylenimine (PEI)/DNA-betagal vector to investigate the specificity and efficiency of immuno-targeting prostate-specific membrane antigen (PSMA). Coupling of the PSMA-specific monoclonal antibody, J591, to the vector was facilitated via the high-affinity interaction between phenyl(di)boronic acid and salicylhydroxamic acid molecules. Highly efficient gene delivery by this prostate cancer (PCA)-targeted J591/polyethylene glycol (PEG)/PEI/DNA-betagal vector was demonstrated in PSMA-positive cells relative to controls, resulting in significant growth inhibition in vitro when the J591/PEG/PEI/DNA-p53 was used. Competition with free antibody resulted in about 90% reduction in both J591 internalization and betagal gene delivery, indicating specificity for PSMA-positive cells. More importantly, testing the efficiency of the J591/PEG/PEI/DNA-betagal targeting vector in an orthotopic PCA model in nude mice resulted in up to a 20-fold increase in gene delivery over the untargeted vector controls. The in vivo organ distribution profile also revealed betagal expression predominantly in the tumor, which was more than 1 log higher than the next highest level of expression in the lung. Furthermore, with the targeted vector containing the gene for yellow fluorescent protein or biotinylated J591, we further demonstrate in vivo that vector-mediated gene delivery is specific for both tumor cells and tumor-associated neovasculature in PSMA-positive tumors. These results suggest the potential for further optimization of this novel vector in the context of therapeutic gene delivery.     

Plasmid-based gene delivery to target tissues in vivo: the intravascular approach

Over the past several years, significant progress has been made in the development of non-viral methodologies that can effectively deliver genes to target tissues in vivo. One of the most surprising successes has been the discovery that naked plasmid DNA (pDNA) can be delivered into tissues such as liver and muscle with high efficiency using the vascular system. The key breakthrough involved the realization that pDNA could be injected rapidly into blood vessels (using increased volumes) in a manner that facilitates extravasation of the DNA solution outside the blood vessel wall. The extravasation process places the DNA in contact with the plasma membranes of the underlying parenchymal cells of the target organ. This intravascular delivery technique, termed 'hydrodynamic delivery', has become established as the primary non-viral methodology for delivering pDNA expression constructs to target tissues in vivo. This review highlights many of the most recent studies in which increased volume/rapid injection procedures have been used. These include studies in which the technology was used as a new and powerful tool to address in vivo gene expression questions, as well as numerous studies that were designed to better understand or improve the methodology. It is these scientific efforts that have served to fuel the development of this delivery technology from simply an interesting phenomenon to a highly useful and broadly used gene delivery methodology.     

Using magnetic forces to enhance non-viral gene transfer to airway epithelium in vivo

We have assessed whether magnetic forces (magnetofection) can enhance non-viral gene transfer to the airways. TransMAG(PEI), a superparamagnetic particle was coupled to Lipofectamine 2000 or cationic lipid 67 (GL67)/plasmid DNA (pDNA) liposome complexes. In vitro transfection with these formulations resulted in approximately 300- and 30-fold increase in reporter gene expression, respectively, after exposure to a magnetic field, but only at suboptimal pDNA concentrations. Because GL67 has been formulated for in vivo use, we next assessed TransMAG(PEI) in the murine nasal epithelium in vivo, and compared this to naked pDNA. At the concentrations required for in vivo experiments, precipitation of magnetic complexes was seen. After extensive optimization, addition of non-precipitated magnetic particles resulted in approximately seven- and 90-fold decrease in gene expression for naked pDNA and GL67/pDNA liposome complexes, respectively, compared to non-magnetic particles. Thus, whereas exposure to a magnetic field improved in vitro transfection efficiency, translation to the in vivo setting remains difficult.     

PEGylated polyethylenimine for in vivo local gene delivery based on lipiodolized emulsion system.

Polyethylenimine (PEI) is one of the most efficient vectors for non-viral gene delivery, whereas its poor transfection activity, compared to viral vectors, and cytotoxicity need to be improved for in vivo applications. In this study, we prepared two PEI conjugates with 6 and 10 wt.% of poly(ethylene glycol) (PEG) grafts (referred to PEI-PEG-6 and PEI-PEG-10, respectively) in order to investigate the effects of PEGylation on cytotoxicity and transfection activity in vitro. In addition, their suitability as vectors for local gene delivery in vivo was assessed by injecting lipiodolized emulsions containing polymer/DNA complexes into the femoral artery of Sprague-Dawley (SD) rats, occluded by a surgical suture to block inflow of the blood to the leg. Both PEGylated PEIs showed significantly lower cytotoxicity and higher transfection activity in COS-1 cells than PEI taken as a control; in particular, PEI-PEG-10 produced the most promising results. The stable water-in-oil emulsion, composed of aqueous domains containing the complexes and lipiodol as an oil phase, was formed in the presence of a hydrogenated castor oil. From in vivo experiments, it was found that all the complexes, dispersed in the lipiodolized emulsion, delivered effectively gene to muscle, surrounding the injection site, rather than other organs such as liver, spleen, kidney, heart and lung. The in vivo transfection activity of PEI-PEG-10 was 3-folds higher in muscle than that of PEI. Based on these results, it can be concluded that PEGylated PEIs (based on the lipiodolized emulsion system) hold a promising potential for local gene delivery in vivo.     

Optical imaging of transferrin targeted PEI/DNA complexes in living subjects

Noninvasive optical bioluminescence imaging systems are important tools for evaluating gene expression in vivo for study of individual and temporal variation in a living animal. In this report, we demonstrate that expression of the firefly luciferase reporter gene (fl) delivered by transferrin (Tf) targeted polyethylenimine (PEI) complexes with, or without, poly(ethylene glycol) (PEG) modifications can be imaged in living A/J mice bearing N2A tumors using a cooled charged coupled device (CCD) camera. Tf-PEI-PEG, Tf-PEI, and PEI (positive control) complexes were tail-vein injected and mice were imaged at 5, 24, 48, and 72 h after complex injection. After imaging, the organs were analyzed ex vivo for firefly luciferase protein (FL) activity. The Tf and PEG modified formulations show significantly (P<0.05) higher FL activity in vivo and ex vivo at the tumor as compared to other organs, including the lungs (a site of high expression with PEI, the positive control). Furthermore, the in vivo bioluminescent signal correlated well (R(2)=0.83) with ex vivo FL activity. These data support that noninvasive imaging of fl reporter expression can be used to monitor the specificity of Tf-PEI and Tf-PEI-PEG polyplex targeting of N2A tumors in A/J mice.     

Synthesis and application of a non-viral gene delivery system for immunogene therapy of cancer.

The synthesis and gene delivery application of a novel lipopolymer, PEG-PEI-CHOL (PPC), is described. PPC is composed of a low molecular weight branched polyethylenimine (PEI) covalently linked with functional groups methoxypolyethyleneglycol (PEG) and cholesterol (CHOL). The potential utility of PPC as a gene delivery polymer was evaluated by showing its ability to form stable nanocomplexes with DNA, protect DNA from degradation by DNase and mediate gene transfer in vitro and in vivo in solid tumors. The ratio of PEG/PEI/CHOL and nitrogen to phosphate (Polymer/DNA) was optimized for physico-chemical properties and gene delivery efficiency of PPC/DNA complexes. The gene therapy application of the polymer was shown following administration of a murine IL-12 plasmid (pmIL-12) formulated with PPC into tumors in mice which resulted in significant inhibition of tumor growth. The inhibitory effects of pmIL-12/PPC were enhanced when combined with specific chemotherapeutic agents, demonstrating the potential usefulness of pIL-12/PPC as an adjuvant therapy for cancer treatment.     

Optimization of regional intraarterial naked DNA-mediated transgene delivery to skeletal muscles in a large animal model.

Effective gene therapy for muscular dystrophy will likely require intravascular administration. Although plasmid DNA (pDNA) contained within a large volume and rapidly infused into a major artery can achieve gene transfer within downstream muscles, this is associated with substantial muscle edema. Here we hypothesized that excessive edema-related increases in intramuscular pressure (IM pressure) developed during intraarterial pDNA injections could hinder successful gene delivery. Accordingly, we monitored IM pressure during injection of pDNA carrying a LacZ transgene into the femoral artery of rats and pigs. Large variations in IM pressure were found between different muscles. There was a significant inverse relationship between IM pressure and the subsequent level of gene transfer to muscle. Modification of the injection protocol to reduce IM pressure led to greatly increased pDNA-mediated gene expression and reduced muscle damage in pigs. Under the most optimized conditions, average transfection within eight different muscles of the pig hind limb amounted to 22% of all fibers, attaining a maximum of 60% in the gastrocnemius muscle. We conclude that IM pressure monitoring is a simple and useful procedure, which can be applied in both small and large animals to help optimize pDNA-mediated gene transfer to skeletal muscles by the intraarterial route.